The DNA of all organisms undergoes persistent damage that can result in genetic mutations or epigenetic changes unless the damage is correctly repaired. The modified base, 5-methylcytosine (5mC), can undergo both deamination and oxidation resulting in both genetic mutations and epigenetic perturbation. We have demonstrated that the oxidation damage product, 5-hydroxymethylcytosine (5hmC), inhibits the binding of proteins that selectively bind to methylated DNA and that the maintenance methyltransferase DNMT1 does not recognize 5hmC when methylating DNA following DNA replication. Therefore, the formation of 5hmC could heritably alter epigenetic patterns in replicating cells. Emerging evidence indicates, however, that the conversion of 5mC to 5hmC could also be part of an enzymatic epigenetic reprogramming pathway essential for stem cell differentiation and that 5hmC is absent from most if not all human cancer cells. In this application, we propose a series of studies to further understand this putative DNA demethylation pathway in human neural stem cells, mouse embryonic stem cells and in a series of human cancer stem cells and established cell lines. We wish to understand how DNA demethylation relies upon DNA damage repair pathways, and how DNA damage repair and DNA demethylation might become entangled. In the first aim, we propose a novel stable isotope labeling method that will allow us to follow the dynamic processes of DNA replication, methylation and hydroxylation and excision of possible intermediates by the base excision repair pathway. In the second aim, we propose to use a series of innovative new methods to identify and quantify enzymatic activities that might act on pathway intermediates, further defining as yet unknown parts of this pathway. In the third aim, we propose some novel mass spectrometry approaches to identify proteins that bind to intermediates of the pathway and to measure how specific DNA intermediates, including 5hmC, affect the specificity and magnitude of the DNA-protein interactions. The proposed studies will allow examination of this important demethylation pathway at an unprecedented level. The information obtained is essential for understanding the biology of normal stem cells within the context of regenerative medicine, and understanding how the pathway becomes defective in human cancer cells could provide new insights into novel targeted chemotherapy.